Metastatic breast cancer is a frequently fatal disease that requires new therapy. Breast cancer is the most common cause of cancer death in women worldwide. There were 39,600 estimated breast cancer deaths in the U.S. in 2002 (Hindle, W. 2002. Breast cancer: introduction. [Review] [33 refs]. Clinical Obstetrics & Gynecology 45, 738-745).
Therapy for metastatic breast cancer is inadequate. In most cases, metastatic breast cancer cannot be cured and current medical therapy is merely palliative (Chew, H. K. 2002. Medical management of breast cancer: today and tomorrow. [Review] [75 refs]. Cancer Biotherapy & Radiopharmaceuticals 17, 137-149; Danova, M., Porta, C., Ferrari, S., and Riccardi, A. 2001. Strategies of medical treatment for metastatic breast cancer (Review). [Review] [64 refs]. International Journal of Oncology 19, 733-739. )
The survival of patients with metastatic disease has not consistently improved over the past decade. The incidence of breast cancer is rising and may increase further with the widespread use of estrogen replacement therapy in post-menopausal women. Despite the advances in early detection conferred by mammography, prevention of breast cancer is presently not feasible to any significant extent.
Weekly treatment with a humanized antibody to the HER2/neu receptor called Herceptin® (Trastuzumab) has been promising as both as a single agent and in combination with standard chemotherapy in patients with HER2/neu over-expressing metastatic breast cancer. (Pegram, M. D., Lipton, A., Hayes, D. F., Weber, B. L., Baselga, J. M., Tripathy, D., Baly, D., Baughman, S. A., Twaddell, T., Glaspy, J. A., and Slamon, D. J. 1998. PHASE II STUDY OF RECEPTOR-ENHANCED CHEMOSENSITIVITY USING RECOMBINANT HUMANIZED ANTI-P185(HER2/NEU) MONOCLONAL ANTIBODY PLUS CISPLATIN IN PATIENTS WITH HER2/NEU-OVEREXPRESSING METASTATIC BREAST CANCER REFRACTORY TO CHEMOTHERAPY TREATMENT. Journal of Clinical Oncology 16, 2659-2671; Vogel, C. L., Cobleigh, M. A., Tripathy, D., Gutheil, J. C., Harris, L. N., Fehrenbacher, L., Slamon, D. J., Murphy, M., Novotny, W. F., Burchmore, M., Shak, S., Stewart, S. J., and Press, M. 2002. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. Journal of Clinical Oncology 20, 719-726; Slamon, D. J., Leyland-Jones, B., Shak, S., Fuchs, H., Paton, V., Bajamonde, A., Fleming, T., Eiermann, W., Wolter, J., Pegram, M., Baselga, J., and Norton, L. 2001. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. [comment]. New England Journal of Medicine 344, 783-792; Cobleigh, M. A., Vogel, C. L., Tripathy, D., Robert, N. J., Scholl, S., Fehrenbacher, L., Wolter, J. M., Paton, V., Shak, S., Lieberman, G., and Slamon, D. J. 1999. Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. Journal of Clinical Oncology 17, 2639-2648). Gene amplification of Her2/neu has been noted in 10-40% of primary human breast, ovarian, cervical, endometrial, lung, and pancreatic cancers and is an independent and strong predictor of poor prognosis. (Kern, J. A., Schwartz, D. A., Nordberg, J. E., Weiner, D. B., Greene, M. I., Torney, L., and Robinson, R. A. 1990. p185 neu expression in human lung adenocarcinomas predicts shortened survival. Cancer Research 50, 5184-5187; Press, M. F., Cordon-Cardo, C., and Slamon, D. J. 1990. Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene 5, 953-962; Press, M. F., Pike, M. C., Hung, G., Zhou, J. Y., Ma, Y., George, J., Dietz-Band, J., James, W., Slamon, D. J., Batsakis, J. G., and et al. 1994. Amplification and overexpression of HER-2/neu in carcinomas of the salivary gland: correlation with poor prognosis. Cancer Research 54, 5675-5682; Niehans, G. A., Singleton, T. P., Dykoski, D., and Kiang, D. T. 1993. Stability of HER-2/neu expression over time and at multiple metastatic sites. Journal of the National Cancer Institute 85, 1230-1235).
[Viruses and viral vectors that kill specific cells are being designed for cancer therapy (Ring, C. J., 2002. Cytolytic viruses as potential anti-cancer agents. J. Gen. Virol. 83, 491-502; Russell, S. J., 1994. Replicating vectors for cancer therapy: a question of strategy. Semin. Cancer Biol. 5, 437-443; Wildner, O., 2001. Oncolytic viruses as therapeutic agents. Ann. Med. 33, 291-304; Zwiebel, J. A., 2001. Cancer gene and oncolytic virus therapy. Semin. Oncol. 28, 336-343. ) Cytolytic replicating viruses represent a new and fundamentally different approach to cancer therapy. Cytolytic viruses differ from standard chemotherapy and radiation therapy in how they target and kill tumor cells. Targeting is achieved by specific binding of the virus to the cancer cell. Viral cytolysis is not dependent on cell growth or division. Viral delivery to its target can be achieved not only via the bloodstream and lymphatic system, but also by cell to cell transmission, spread through the interstitial fluid both within and between tissue planes and by perineural spread. (Tyler, K L. and Nathanson, N. (2001). Pathogenesis of Viral Infections. In “Fundamental Virology” (D. Knipe and P. Howley, Eds.), pp. 199-244. Lippincott Williams & Wilkins, Philadelphia; Plakhov, I. V., Arlund, E. E., Aoki, C., and Reiss, C. S. 1995. The earliest events in vesicular stomatitis virus infection of the murine olfactory neuroepithelium and entry of the central nervous system. Virology 209, 257-262; Vassalli, J. D., Lombardi, T., Wohlwend, A., Montesano, R., and Orci, L. 1986. Direct cell-to-cell transmission of vesicular stomatitis virus. Journal of Cell Science 85, 125-131). Replicating viruses do not have to be delivered to every tumor cell on the first pass but can spread in waves from the initial site of infection throughout the entire tumor. (Wu, J. T., Byrne, H. M., Kim, D. H., and Wein, L. M. 2001. Modeling and analysis of a virus that replicates selectively in tumor cells. Bulletin of Mathematical Biology 63, 731-768). Virus therapy would be unique among biological and chemotherapies in having the potential to be more effective in solid masses of tumor than in minimal residual disease. Moreover, large tumor deposits may initially shield virus from the host immunologic response, because they are devoid of lymphatic drainage, express few MHC antigens and elaborate locally immunosuppressive products. (Russell, S. J. 1994. Replicating vectors for gene therapy of cancer: risks, limitations and prospects. [Review]. European Journal of Cancer 30A, 1165-1171).
Breast cancer represents an excellent target for a cytolytic virus, because metastatic disease occurs in masses; tumor associated cell surface receptors are known; and breast tissue is not essential. Destruction of all breast tissue, cancerous or non-cancerous, by direct viral cytolysis or indirect host immunologic response is an acceptable outcome where necessary to achieve therapeutic benefit.
Synergy between virus therapy and chemotherapy is possible because: 1) the basis of viral selectivity is not the faster growth rate of tumor cells compared with most normal tissues; 2) viral oncolysis is likely to produce inflammation and neovascularization within the tumor which will promote delivery of chemotherapeutic agents to tumor cells; 3) chemotherapeutic agents will suppress the host immunologic response and prolong the duration of viral spread and oncolysis. Overlapping toxicities between viral therapy and chemotherapy are not expected because the mechanisms of action are so different (Nemunaitis, J., Cunningham, C., Tong, A. W., Post, L., Netto, G., Paulson, A. S., Rich, D., Blackburn, A., Sands, B., Gibson, B., Randlev, B., and Freeman, S. 2003. Pilot trial of intravenous infusion of a replication-selective adenovirus (ONYX-015) in combination with chemotherapy or IL-2 treatment in refractory cancer patients. Cancer Gene Therapy 10, 341-352).
Vesicular Stomatitis Virus (VSV) is an excellent candidate for development as an oncolytic virus, because it is an efficient cell killer that grows and spreads rapidly and yet is safe for human use (de Mattos, C. A., de Mattos, C. C., Rupprecht, C. E., 2001. Rhabdoviruses. In: Knipe, D., Howley, P. (Eds.), Fundamental Virology. Lippincott Williams & Wilkins, Philadelphia, pp. 1245-1277). VSV is endemic in certain human populations, but is not pathogenic. Wild type (wt) VSV has eradicated established tumors in mice when injected intratumorally or intravenously (Balachandran, S., Porosnicu, M., Barber, G. N., 2001. Oncolytic activity of vesicular stomatitis virus is effective against tumors exhibiting aberrant p53, Ras, or myc function and involves the induction of apoptosis. J. Virol. 75, 3474-3479; Fernandez, M., Porosnicu, M., Markovic, D., Barber, G. N., 2002. Genetically engineered vesicular stomatitis virus in gene therapy: application for treatment of malignant disease. J. Virol. 76, 895-904). Selectivity was based on the absence of an interferon response in the tumor cells (Stojdl, D. F., Lichty, B., Knowles, S., Marius, R., Atkins, H., Sonenberg, N., Bell, J. C., 2000. Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. Nat. Med. 6, 821-825).
VSV has many advantages for development as cancer therapy including, most importantly, safety. Although primarily a mild disease of horses, cattle and swine, there are parts of Central America where serologic studies demonstrate that subclinical infection is common in the human population. Use of this agent does not introduce a new virus into the human host and serious illness almost never occurs. VSV is an RNA virus that cannot integrate into the mammalian genome and has no known transforming abilities. It does not produce a persistent infection. A major reason for safety in the humans is that VSV rapidly induces a strong interferon (IFN) response which protects the host (Ring, C. J. 2002. Cytolytic viruses as potential anti-cancer agents. Journal of General Virology 83, 491-502).
VSV is an enveloped negative strand RNA virus with a single surface glycoprotein (gp) called G that fully determines binding of the virus to target cells as well as promoting pH-dependent fusion of the virus envelope with endosome membranes (Rose, J. K., Whitt, M. A., 2001. Rhabdoviridae: the viruses and their replication. In: Knipe, D., Howley, P. (Eds.), Fundamental Virology Lippincott Williams & Wilkins, Philadelphia, pp. 1221-1244). VSV contains only 5 genes and can be created entirely from vectors that express these genes, without effect on viral packaging. The viral genome has the capacity to accommodate additional genetic material. At least two additional transcription units, totaling 4.5 kb, can be added to the genome. Added genes are stably maintained in the genome upon repeated passage (Schnell, M. J., Buonocore, L., Boritz, E., Ghosh, H. P., Chernish, R., and Rose, J. K. 1998. Requirement for a non-specific glycoprotein cytoplasmic domain sequence to drive efficient budding of vesicular stomatitis virus. EMBO Journal 17, 1289-1296; Schnell, M. J., Buonocore, L., Kretzschmar, E., Johnson, E., and Rose, J. K. 1996a. Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles. Proceedings of the National Academy of Sciences of the United States of America 93, 11359-11365; Schnell, M. J., Buonocore, L., Whitt, M. A., and Rose, J. K. 1996b. The minimal conserved transcription stop-start signal promotes stable expression of a foreign gene in vesicular stomatitis virus. Journal of Virology 70, 2318-2323; Kahn, J. S., Schnell, M. J., Buonocore, L., and Rose, J. K. 1999. Recombinant vesicular stomatitis virus expressing respiratory syncytial virus (RSV) glycoproteins: RSV fusion protein can mediate infection and cell fusion. Virology 254, 81-91).
VSV infection also elicits strong humoral and cellular immune responses and evokes an inflammatory response at the site of infection that includes macrophages, neutrophils and lymphocytes. VSV incorporates portions of the cellular plasma membrane into its envelope and can cause the immune system to react to these antigens. Immunization with VSV grown in myelin basic protein (MBP) expressing cell cultures evoked a T cell response to the “self” MBP protein (Rott, O., Herzog, S., and Cash, E. 1994. Autoimmunity caused by host cell protein-containing viruses. Medical Microbiology & Immunology 183, 195-204).
VSV has also been demonstrated to be a potent oncolytic virus. It kills any tumor cell that it infects within hours and affects both dividing and non-dividing cells. Studies of subcutaneous and pulmonary tumors in mice demonstrated that wild type (wt) VSV administered directly into subcutaneous tumor at a dose of 2×107 PFU (plaque forming units) or IV at a dose of 5×106 PFU achieved tumor regression but did not produce replicating infections in body organs (Fernandez, M., Porosnicu, M., Markovic, D., and Barber, G. N. 2002. Genetically engineered vesicular stomatitis virus in gene therapy: application for treatment of malignant disease. Journal of Virology 76, 895-904). Many tumor cell types have lost responsiveness to IFN and are therefore very sensitive to killing by VSV, making this virus an excellent candidate for cancer therapy (Stojdl, D. F., Lichty, B., Knowles, S., Marius, R., Atkins, H., Sonenberg, N., and Bell, J. C. 2000. Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. Nature Medicine 6, 821-825; Stojdl, D. F., Lichty, B. D., tenOever, B. R., Paterson, J. M., Power, A. T., Knowles, S., Marius, R., Reynard, J., Poliquin, L., Atkins, H., Brown, E. G., Durbin, R. K., Durbin, J. E., Hiscott, J., and Bell, J. C. 2003a. VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents. Cancer Cell 4, 263-275; Stojdl, D. F., Lichty, B. D., tenOever, B. R., Paterson, J. M., Power, A. T., Knowles, S., Marius, R., Reynard, J., Poliquin, L., Atkins, H., Brown, E. G., Durbin, R. K., Durbin, J. E., Hiscott, J., and Bell, J. C. 2003b. VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents. Cancer Cell 4, 263-275).
The present inventors have previously reported the development of a recombinant VSV whose only surface glycoprotein (gp) was a Sindbis virus (SV) gp, called Sindbis-ZZ, which could be targeted to breast cancer cells (Bergman et al. Vesicular stomatitis virus expressing a chimeric Sindbis glycoprotein containing an Fc antibody binding domain targets to Her2/neu overexpressing breast cancer cells. Virology. Nov. 25, 2003;316(2):337-47. ) The cellular receptor for G is ubiquitous and VSV promiscuously infects most cell types. The surface gp of SV consists of an E1 fusion protein and an E2 binding protein. Deletion of amino acids 72 and 73 within E2 reduces binding and infectivity of the virus >90% (Dubuisson, J., Rice, C. M., 1993. Sindbis virus attachment: isolation and characterization of mutants with impaired binding to vertebrate cells. J. Virol. 67, 3363-3374). The Sindbis gp gene was further modified at this site by others to encode two synthetic immunoglobulin G (IgG) Fc-binding domains called ZZ derived from protein A of the Staphylococcus aureus spa gene (Ohno, K., Sawai, K., Iijima, Y., Levin, B., Meruelo, D., 1997. Cell-specific targeting of Sindbis virus vectors displaying IgG-binding domains of protein A. Nat. Biotechnol. 15, 763-767; Morizono, K., Bristol, G., Xie, Y. M., Kung, S. K., Chen, I. S., 2001. Antibody-directed targeting of retroviral vectors via cell surface antigens. J. Virol. 75, 8016-8020; Sawai, K., Meruelo, D., 1998. Cell-specific transfection of choriocarcinoma cells by using Sindbis virus hCG expressing chimeric vector. Biochem. Biophys. Res. Commun. 248, 315-323). Sindbis viruses and retroviruses expressing this ZZ-modified gp could be targeted to specific cells by the addition of antibody. The inventors incorporated this glycoprotein gene into the VSV genome and made a VSV that expressed this modified Sindbis gp and not the native VSV G gp. Genetic engineering has previously been developed to create VSV from plasmid components (Schnell, M. J., Buonocore, L., Kretzschmar, E., Johnson, E., Rose, J. K., 1996. Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles. Proc. Natl. Acad. Sci. U.S.A. 93, 11359-11365). The inventors showed that VSV recombinant virus and pseudotype virus expressing the Sindbis ZZ gp could be targeted to Her2/neu expressing breast cancer cells using antibody to Her2/neu.
Targeting of the VSV containing Sindbis-ZZ however was inefficient, since its mechanism required the intermediate binding of a cell-specific antibody with a non-specific antibody binding site expressed on the viral surface. In vivo competition for the non-specific antibody binding site will include the large pool of host IgG antibodies. In addition, selective adaptation of this virus on targeted cells was difficult, because each new generation of virus required additional antibody to allow binding and infection of the next round of cells.
Previous attempts to target viruses to cancer cells using single chain antibodies (SCA) have also been limited due to low titer. Previous reports using SCA to target retroviruses, adeno-associated virus (AAV) and attenuated measles virus (MV) produced viral titers of about 1×105/ml (Jiang et al., 1998; Khare et al., 2001; Marin et al., 1996; Martin et al., 2003; Yang et al., 1998). Also, the SCA gene was not incorporated into the retroviral genome and the viruses could not replicate. In addition, these viruses cannot be used directly to kill tumor cells because they are not cytolytic. For MV, the titers against specific cells were 6×104-6×105/ml and in addition native MV binding was not at all attenuated. Rather, the tropism of the virus was extended to cells not normally infected by MV. Unlike retroviruses, however, these targeted MV were replication competent and cytolytic (Bucheit et al., 2003; Hammond et al., 2001; Peng et al., 2003).
Although VSV is a potent oncolytic virus, wild type VSV can cause significant morbidity if the virus enters the brain of animals. The possibility of such movement into the brain becomes a significant barrier to its practical application for therapeutic purposes.
Thus, there remains a need in the art for viruses, compositions and methods which enable safe, efficient and effective use of these viruses in the treatment of disorders and diseases, such as breast cancer.